Unique peptidic agonists of a juvenile hormone receptor with species-specific effects on insect development and reproduction
Edited by Lynn Riddiford, University of Washington, Friday Harbor, WA; received September 15, 2022; accepted October 20, 2022
Significance
Synthetic mimics of the insect juvenile hormone (JH) exemplify biorational pesticides. Unique peptidic juvenoids with target-species specific effects, were discovered in the early 1970s. However, these promising compounds were abandoned with their mode of action never addressed. Our study shows that peptidic juvenoids act through the canonical JH receptor signaling to affect development and reproduction of the target insect. Our newly synthesized peptidic derivatives outperform established JH mimics by two orders of magnitude while retaining their target species selectivity. We further propose an advanced structural model for interaction of these highly potent agonists with the JH receptor. These results unveil the potential of peptidic juvenoids as agents for precisely targeted and sustainable insect control.
Abstract
Juvenile hormones (JHs) control insect metamorphosis and reproduction. JHs act through a receptor complex consisting of methoprene-tolerant (Met) and taiman (Tai) proteins to induce transcription of specific genes. Among chemically diverse synthetic JH mimics (juvenoids), some of which serve as insecticides, unique peptidic juvenoids stand out as being highly potent yet exquisitely selective to a specific family of true bugs. Their mode of action is unknown. Here we demonstrate that, like established JH receptor agonists, peptidic juvenoids act upon the JHR Met to halt metamorphosis in larvae of the linden bug, Pyrrhocoris apterus. Peptidic juvenoids induced ligand-dependent dimerization between Met and Tai proteins from P. apterus but, consistent with their selectivity, not from other insects. A cell-based split-luciferase system revealed that the Met–Tai complex assembled within minutes of agonist presence. To explore the potential of juvenoid peptides, we synthesized 120 new derivatives and tested them in Met–Tai interaction assays. While many substituents led to loss of activity, improved derivatives active at sub-nanomolar range outperformed hitherto existing peptidic and classical juvenoids including fenoxycarb. Their potency in inducing Met–Tai interaction corresponded with the capacity to block metamorphosis in P. apterus larvae and to stimulate oogenesis in reproductively arrested adult females. Molecular modeling demonstrated that the high potency correlates with high affinity. This is a result of malleability of the ligand-binding pocket of P. apterus Met that allows larger peptidic ligands to maximize their contact surface. Our data establish peptidic juvenoids as highly potent and species-selective novel JHR agonists.
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Juvenile hormones (JHs) comprise a group of sesquiterpenoids modified by one or two epoxide rings (1–3). JH was originally recognized by virtue of its capacity to maintain insect larvae in the juvenile state prior to metamorphosis (4–7). In adult insects, JH signaling regulates reproduction, often by stimulating vitellogenesis in females (8, 9). The effects of JH are mediated by an intracellular receptor that consists of transcription factors belonging to the basic helix-loop-helix/Per-Arnt-Sim (bHLH-PAS) family (see ref. 10, for a recent review). A functional JH receptor (JHR) consists of a heterodimer of proteins originally described as methoprene-tolerant (Met) (11) and taiman (Tai) (12), which interact in the presence of an agonist ligand bound to Met (13–16). Upon ligand-induced dimerization, the Met–Tai complex binds cis-regulatory JH-response elements and activates transcription of genes such as the suppressor of metamorphosis, Krüppel-homolog 1 (Kr-h1) (14, 15, 17–19).
Besides the natural insect JHs, numerous synthetic compounds have been found to mimic JH effects (20–22). When ectopically applied to pre-adult stages, such as final-instar larvae and pupae of many insect species, JH or its mimics (juvenoids) disrupt metamorphosis by perpetuating the juvenile status even beyond the following molt (5). In insects such as the fly Drosophila melanogaster, juvenoid treatment leads to death during metamorphosis (23, 24). The potential utility of JH mimics was recognized early during JH research (25), and some of these compounds found use as the first insecticides of the insect growth regulator (IGR) class (21, 22, 26). Following identification of Met as an essential component of the JHR, established juvenoid IGRs including methoprene, pyriproxyfen, and fenoxycarb have been shown to act as JHR agonists via high-affinity, competitive binding to Met orthologs of diverse species (13, 27–29).
A peptidic molecule with juvenoid activity was inspired by the structure of a plant-derived JH mimic juvabione (30), the so-called paper factor originating from the balsam fir (31). Like juvabione, these peptidic derivatives of p-aminobenzoate had a systemic anti-metamorphic effect on true bugs of the family Pyrrhocoridae, namely on the genera Dysdercus and Pyrrhocoris (32–34). By the early 1970s, several peptidic juvenoids (PJs) were developed into highly potent, species-selective JH mimics (35). However, the potential of PJs as agents for insect control and their mode of action have remained unexplored, possibly owing to a traditional preference for compounds active in a broad spectrum of species.
The linden bug, Pyrrhocoris apterus, is a tractable model for assessing JH activity on both development and reproduction. When exposed to a JH mimic such as methoprene, final-instar larvae fail to form adults and instead molt to an extra larval instar in a manner dependent on Met (36). Under short daylight, adult P. apterus females enter reproductive diapause, during which neither endogenous JH nor vitellogenic oocytes are produced (37, 38). Administration of methoprene whose action is mediated by the JHR genes Met and tai overrides the photoperiod-dependent diapause and induces oogenesis in formerly non-reproductive females (37, 38).
Recognizing the potential of the PJs as uniquely selective juvenoid IGRs, we set out to address the issue of the molecular action of these peptides. We have ascertained that the original, active PJ compound (35) exerts the anti-metamorphic effect on P. apterus larvae through the JHR protein Met. Active PJs stimulate interaction between the Met and Tai JHR proteins from P. apterus but not between their orthologs from non-target species. Our new PJ derivatives by far surpass JH activities of any formerly described PJ compounds and of previously established JHR agonists. A structural basis for the improved agonist activity is proposed.
Results and Discussion
Peptidic Juvenoid Blocks Metamorphosis through the JHR Met.
Without ectopic hormonal intervention, final (fifth instar) larvae (L5) of P. apterus express very little Kr-h1 mRNA (39) and hence molt to adults with developed genitalia, wings, and a specific color pattern. In contrast, L5 individuals treated with the JHR agonist methoprene strongly express Kr-h1 and molt to giant L6 larvae (36) (SI Appendix, Fig. S1).
Given the distinct chemistry of PJs (SI Appendix, Fig. S2), it was critical to determine whether these peptides exert their anti-metamorphic effect through the recognized JH signaling pathway. To this end, we synthetized one of the original, highly active compounds, ethyl pivaloyl-l-alanyl-p-aminobenzoate (35), here renamed ethyl (S)-4-(2-pivalamidopropanamido)benzoate and abbreviated PJ-1 (Fig. 1A and SI Appendix, Materials and Methods). In contrast to controls, P. apterus L5 larvae treated with PJ-1 ectopically expressed high levels of Kr-h1 mRNA (Fig. 1B) before molting to the supernumerary L6 instar and thus phenocopied the effect of methoprene (Fig. 1C and Table 1). Importantly, neither PJ-1 nor methoprene induced the extra larval molt in animals that had been previously subjected to Met knockdown (Fig. 1C and Table 1). These data indicate that, similar to formerly established JHR agonists, PJs achieve their anti-metamorphic effect through JH signaling, via Met-dependent activation of the Kr-h1 gene.
Fig. 1.
PJ-1 Induces Interaction between P. apterus Met and Tai Proteins.
To assess agonist activities of natural JHs and synthetic juvenoids at the molecular level, we utilized two methods that measure ligand-induced dimerization of Met with Tai: a previously established two-hybrid assay (28, 40) and the NanoLuc binary technology (NanoBiT) (41) which we have adopted for JHR (this study). Both assays are conducted in mammalian cell lines (see Materials and Methods for details).
In the two-hybrid assay, dimerization of the P. apterus Tai (PaTai) and Met (PaMet) proteins fused to the Gal4 DNA-binding and the VP16 transactivation domains, respectively, results in expression of a UAS-driven luciferase reporter. The interaction between PaMet and PaTai was induced by the native JH of the linden bug, JH III skipped bisepoxide (JHSB3) (38, 42), and by the synthetic JHR agonists methoprene and fenoxycarb (Fig. 2A). Agonist structures are depicted in SI Appendix, Fig. S2. PaMet and PaTai also interacted in the presence of hormones native to other insects, JH I and JH III (SI Appendix, Fig. S3). PJ-1 stimulated PaMet-PaTai dimerization with an effective concentration (EC50) of 4.6 ± 2.5 nM (n = 5), whereas its stereoisomer PJ-1-D with d-alanyl in the central position was inactive (Fig. 2A). These results were consistent with the anti-metamorphic activity of PJ-1 reported for P. apterus L5 larvae (EC50 = 0.4–10 ng per animal) and lack of such activity in the case of PJ-1-D (35, 43) (SI Appendix, Fig. S4A). The potency of PJ-1 in the two-hybrid assay was slightly below that determined for fenoxycarb (1.1 ± 0.4 nM; n = 5) but exceeded those of methoprene (18.0 ± 5.2 nM; n = 3) or JHSB3 (67.8 ± 36.2 nM; n = 3) (Fig. 2A). The comparatively lower potency of JHSB3 might have been partly due to impurity of the available JHSB3 sample (SI Appendix, Fig. S5).
Fig. 2.
We next examined whether the effect of PJ-1 on Met–Tai dimerization was specific and likely dependent on the ligand-binding capacity of Met. Several amino acid residues within the ligand-binding pocket of Met have been identified whose mutations block binding of JH III to Met proteins from diverse insect species (13, 18, 27, 44). Thus, we replaced a conserved threonine at position 274 of P. apterus Met (SI Appendix, Fig. S6) with the bulkier tyrosine residue, a mutation known to preclude ligand binding. In contrast to the functional (WT) Met protein, the T274Y variant showed no agonist-dependent interaction with Tai in response to PJ-1, JH I, or methoprene (Fig. 2B). Another mutation that markedly reduced the ability of agonists including PJ-1 to induce Met–Tai interaction affects tryptophan 272 of P. apterus Met (Fig. 2B). Met orthologs of insects other than true bugs (Heteroptera) harbor tyrosine at the corresponding position (SI Appendix, Fig. S6). Of note, the hydroxyl group of this tyrosine residue may form a hydrogen bond that contributes to binding of either JH III or a synthetic agonist to Met orthologs of Drosohila melanogaster (28, 45). The loss of agonist-dependent interaction between the mutated Met variants and Tai suggests that a ligand-binding activity of P. apterus Met is required for PJ-1 action.
To corroborate our results in a system independent of transcriptional induction, we used the NanoBiT technique, in which interaction of two proteins, fused to complementary fragments of the NanoLuc luciferase, reconstitutes the active luciferase (SI Appendix, Fig. S7). Luminescence is recorded in live cells, previously permeated with a furimazine-containing substrate. Interaction between PaMet and PaTai was induced by JHR agonists including JHSB3, JH III, and fenoxycarb (Fig. 2 C and D). The signal culminated by 10 min and plateaued thereafter. Similar profiles resulted whether PaMet was fused to the small and PaTai to the large NanoLuc fragment or viceversa (SI Appendix, Fig. S7), indicating that both combinations were functionally equivalent. At 100 nM, PJ-1 was similarly active as fenoxycarb, whereas the PJ-1-D stereoisomer was inactive (Fig. 2D). The effect of PJ-1 was dose-dependent in terms of both the speed of the response and the maximum activity attained (Fig. 2E). These data show that exposure of cells expressing PaMet and PaTai to JHR agonists, including peptidic juvenoids, triggers a rapid assembly of the JHR complex.
Selective Action of PJ-1 on JHR from P. apterus.
Several peptidic juvenoids including PJ-1 have been known to affect pyrrhocorid bugs but not other insects such as beetles (20, 33). To test whether the selective anti-metamorphic effect of PJ-1 on P. apterus is reflected at the molecular level, we employed the two-hybrid system with Met and Tai orthologs from additional insect species, namely the beetle Tribolium castaneum, the mosquito Aedes aegypti, the termite Prorhinotermes simplex, and D. melanogaster. While the JHR protein pairs from all five species interacted in the presence of the universal JHR agonist fenoxycarb, only the PaMet-PaTai complex formed in response to PJ-1 (Fig. 3A). The selectivity of PJ-1 action was further supported through the NanoBiT approach. While the JHR proteins from T. castaneum (TcMet and TcTai) assembled in response to JH III or fenoxycarb, no interaction was detected upon incubation with PJ-1 (Fig. 3B).
Fig. 3.
New PJs with Improved Potency and Effect on Metamorphosis and Reproduction.
Several PJ compounds with variable anti-metamorphic activities on pyrrhocorid bugs have been described (20, 33, 35). With the aim to expand the repertoire of active PJs and to investigate whether their potency could further be improved, we modified the prototype PJ structure with diverse substituents at the N-terminal side chain (R3), the central amino acid (R2) and, to a limited extent, the C-terminal ester (R1). The syntheses yielded 120 derivatives in addition to PJ-1. Activity of selected PJs was initially tested using a manual mode of the two-hybrid assay based in Chinese hamster ovary (CHO) cells (Fig. 4A). The chemical structures of all PJs are shown in SI Appendix, Materials and Methods; selected examples are also depicted in Fig. 4B and Table 2. While certain modifications led to partial or complete loss of activity, others resulted in molecules outperforming the original PJ-1 in the two-hybrid assay (Fig. 4A). This was corroborated in the NanoBiT assay for PJ-105, which was active at a 10 pM concentration, i.e., hundred-fold lower than that required for an appreciable effect of PJ-1 (Fig. 2E).
Fig. 4.
Table 2.
| Phenotype† | ||||||
|---|---|---|---|---|---|---|
| PJ* | Structure | L6 | Adultoid | Adult | Kr-h1‡ (%) | Met–Tai§ EC50 [nM] |
| Solvent | - | 0 | 0 | 20 | 3 ± 2 | - |
| PJ-2 |  | 0 | 0 | 11 | 4 ± 1 | n.a. |
| PJ-37 |  | 0 | 0 | 13 | 13 ± 9 | 20,000 |
| PJ-69 |  | 0 | 7 | 6 | 48 ± 8 | 8,100 |
| PJ-36 |  | 20 | 9 | 0 | 45 ± 5 | 140 |
| PJ-53 |  | 3 | 8 | 1 | 49 ± 15 | 59 |
| PJ-22 |  | 13 | 0 | 0 | 118 ± 23 | 21 |
| PJ-10 |  | 12 | 1 | 0 | 85 ± 12 | 4.7 |
| PJ-1 |  | 13 | 0 | 0 | 116 ± 11 | 2.4 |
| PJ-52 |  | 13 | 0 | 0 | 81 ± 8 | 0.59 |
| PJ-74 |  | 13 | 0 | 0 | 117 ± 26 | 0.32 |
| PJ-103 |  | 26 | 0 | 0 | 106 ± 33 | 0.13 |
| PJ-101 |  | 9 | 0 | 0 | 126 ± 23 | 0.12 |
| PJ-105 |  | 13 | 0 | 0 | 128 ± 22 | 0.03 |
*
PJs were topically applied (0.6 nmol per specimen) to early L5 larvae of P. apterus.
†
Examples of representative phenotypes are shown in Fig. 4B for selected PJs.
‡
Kr-h1 mRNA level (mean ± SD, n = 3) relative to that induced with methoprene (=100%).
§
Average of NLuc and FLuc activities in two-hybrid assays from Dataset S1.
To provide a precise quantitative evaluation of dose responses for all individual PJ compounds, we upgraded the two-hybrid assay to an automated mode (see SI Appendix, Materials and Methods for details). This mode employed a clonal human osteosarcoma (U2OS) cell line, stably transfected with vectors expressing the PaMet and PaTai proteins, plus a coincidence reporter for simultaneous detection of NanoLuc and firefly luciferase activities (SI Appendix, Fig. S8). The EC50 values and ranking of compounds determined in the automated assay (Dataset S1 and SI Appendix, Fig. S8) were in good agreement with data obtained manually (Fig. 4A).
Based on ranking in the two-hybrid assay, representative PJs were selected for experiments in vivo. At the highest dose tested (0.6 nmol per larva), potent PJs induced the full anti-metamorphic effect (Fig. 4B and Table 2), i.e., molting to a supernumerary larval instar (L6). In contrast, treatment with less potent PJs often led to incomplete adult development, externally visible as an intermediate “adultoid” phenotype (SI Appendix, Fig. S1), whereas PJ derivatives inactive in the two-hybrid assay (such as PJ-2 and PJ-37) did not affect adult morphogenesis at all (Fig. 4B and Table 2). Importantly, the anti-metamorphic activity of PJs rose with their potency in the two-hybrid assay and was reflected in their capacity to induce ectopic expression of Kr-h1 mRNA in P. apterus larvae (Table 2). PJ-101 and PJ-103 were similarly potent, ranking among the top five in the two-hybrid assay (Dataset S1). Accordingly, these compounds exceeded the anti-metamorphic activity of methoprene and PJ-1 (Fig. 4C). PJ-103 is a chlorinated version of PJ-101 (Fig. 4B), and, by reaching an ED50 of 0.4 pmol per larva, it was almost threefold more effective than PJ-101 in blocking metamorphosis (Fig. 4C).
In contrast to the well-documented effect of PJs on metamorphosis (20, 33, 35), their potential to mimic JH in stimulating female reproduction has not been reported. Photoperiod-dependent reproductive diapause in P. apterus provides an opportunity to assess specific JH activity. Exposing females to short daylength shuts JH production down, consequently blocking vitellogenesis (37, 38). When treated with methoprene, formerly diapausing females developed vitellogenic oocytes within 5 d of continuous rearing under short daylength (Fig. 4D). PJ-1, PJ-22, and PJ-101 all had a similar effect when applied to diapausing adult females, which then produced eggs similar to normal reproductive females reared under long daylight (Fig. 4D and SI Appendix, Fig. S4B). In contrast, vitellogenesis was not induced by the inactive PJ-1-D and PJ-37 stereoisomers featuring the d-configuration of the central amino acid (Fig. 4D and SI Appendix, Fig. S4B). These data demonstrate that like other JHR agonists, PJs can specifically override photoperiodic regulation of JH synthesis and induce oogenesis under the conditions that confer reproductive diapause.
Structure-Activity Relationship (SAR).
Accurate ranking of PJs according to their potency in the PaMet-PaTai two-hybrid assay (Dataset S1 and SI Appendix, Fig. S8) enabled us to stratify these compounds based on their structure and agonist activity. Representative structures of PJs were selected to draw SAR models (Tables 3–5).
Table 3.
 | |||
|---|---|---|---|
| R1 | R2 (amino acid) | EC50 [nM]* | |
| PJ-1 | -COOEt | Me (Ala) | 2.4 |
| PJ-22 | -COOMe | Me (Ala) | 21 |
| PJ-44 | -NHCOOMe | Me (Ala) | inactive |
| PJ-101 | -COOMe | tBu (Tle) | 0.12 |
| PJ-105 | -COOEt | tBu (Tle) | 0.03 |
| PJ-108 | -COOiPr | tBu (Tle) | 0.33 |
| PJ-109 | -COOPr | tBu (Tle) | 0.38 |
*
Values from Dataset S1.
Table 4.
 | ||
|---|---|---|
| R2′, R2 (amino acid) | EC50 [nM]* | |
| PJ-22 | H, Me (Ala) | 21 |
| PJ-37 | Me, H (d-Ala) | 20,000 |
| PJ-49 | H, Et (Abu) | 1.9 |
| PJ-64 | Me, Me (Aib) | 8,600 |
| PJ-71 | -CH2,CH2,CH2- | 4,900 |
| PJ-74 | H, iPr (Val) | 0.32 |
| PJ-87 | H, CH2OH (Ser) | 570 |
| PJ-88 | H, CH2-OtBu (Ser(tBu)) | 3,400 |
| PJ-89 | H, iBu (Leu) | 6.4 |
| PJ-93 | H, sBu (Ile) | 2.9 |
| PJ-98 | H, Pr, (Nva) | 0.84 |
| PJ-101 | H, tBu (Tle) | 0.12 |
| PJ-110 | H, CH2-Ph (Phe) | 24 |
*
Values from Dataset S1.
Table 5.
 | |||||||
|---|---|---|---|---|---|---|---|
| R3 | R3 name | EC50 [nM]* | R3 | R3 name | EC50 [nM]* | ||
| PJ-16 |  | methyl | inactive | PJ-25 |  | cyclobutyl | 170 |
| PJ-17 |  | ethyl | inactive | PJ-26 |  | cyclopentyl | 6.7 |
| PJ-18 |  | isopropyl | 920 | PJ-27 |  | cyclo-pentylmethyl | 600 |
| PJ-19 |  | propyl | 1,700 | PJ-28 |  | cyclohexyl | 230 |
| PJ-20 |  | (R,S)-1-methyl propyl | 970 | PJ-29 |  | phenyl | inactive |
| PJ-21 |  | isobutyl | 1,300 | PJ-30 |  | 3-pyridyl | inactive |
| PJ-22 |  | tert-butyl | 21 | PJ-32 |  | 1-chloro-1-methyl-ethyl | 8.3 |
| PJ-23 |  | cyclo-propyl | 1,400 | PJ-33 |  | 1,1-dimethyl-propyl | 20 |
| PJ-24 |  | 3-oxo-cyclobutyl | 46,000 | PJ-36 |  | 2,2-dimethyl-propyl | 140 |
*
Values from Dataset S1.
With the C-terminal (R1) ethyl ester group, PJ-1 was an order of magnitude more potent than the corresponding methyl ester PJ-22 (Table 3). In the series with tert-Leu (R2 = tert-butyl, tBu), the ethyl ester PJ-105 was superior to PJs with both smaller and larger R1 substituents (Table 3), thus confirming early indications from bioassays (46) that ethyl may be preferred at the C terminus of peptidic juvenoids. Presence of the urethane group in PJ-44 (Table 3) and other PJs (Dataset S1) led to complete loss of activity.
At the central amino acid position (R2), PJ activity was enhanced by some bulkier side chains (Table 4 and SI Appendix, Fig. S9). Adding one (PJ-49), two (PJ-74), or three (PJ-101) methyl groups in the amino acid β-position gradually improved the potency by more than two orders of magnitude relative to PJ-22 (Table 4). Other isomeric arrangements of the carbons in the side chain were tolerated (PJ-89, PJ-93, and PJ-98) but were less active than the tBu group of PJ-101. Based on anti-metamorphic activities in P. apterus and D. cingulatus (43, 46), authors of an early attempt to draw a SAR for PJs speculated that a putative binding site accommodates limited extension and branching (Val, Ile) of the R2 substituent, whereas alkyl side chains longer than three carbons do not fit such a site (47).
A polar residue (Ser, PJ-87) substantially reduced activity, and its O-substituted precursor PJ-88 had only micromolar potency. This was consistent with the observation that increasing R2 lipophilicity (cLogP) improved the potency with an optimal cLogP value between 0.75 and 1 (SI Appendix, Fig. S9 A and D). Further, analysis of purely aliphatic R2 substituents revealed that bulkier R2 groups increased agonistic activity and that the binding pocket size limit has probably not been reached (SI Appendix, Fig. S9 B–D). On the other hand, the binding pocket apparently accommodated an aromatic sidechain (Phe) of PJ-110 although the activity was near that of PJ-22 (Table 4). PJs 71 or 64 with disubstituted amino acids had potencies almost five orders of magnitude below that of PJ-101. Therefore, PJ-101 with tBu as the central amino acid remained the most potent analogue of the methyl ester (R1) series, while the corresponding ethyl ester PJ-105 was the most potent compound of the entire peptide library (Dataset S1).
The l-configuration of the central amino acid proved essential for the agonistic activity. This is consistent with a lack of biological activity in PJs bearing d-amino acids as reported earlier (35, 43) and in this study for PJ-1-D and PJ-37 (Fig. 4). Relative to PJ-22, its d-Ala antipode PJ-37 was almost inactive in the two-hybrid assay (Table 4). PJ-64 with two methyl groups exhibited weak activity, indicating that this position only tolerates one substitution of the α-carbon exclusively in the l-configuration (Table 4). These results are in line with the high stereoselectivity of the JHR (28) and provide a molecular basis for the strict preference of the l-configuration observed in bioassays.
The N-terminal position (R3) tolerated diverse groups including linear and ramified aliphatic chains and cyclic aliphatic groups (Table 5 and SI Appendix, Figs. S10 and S11), but introduction of an aromatic cycle (PJ-29 and PJ-30) led to complete loss of activity. Of the modifications to the original tert-butyl at R3 examined here, replacing one methyl group with chlorine (1-chloro-1-methyl-ethyl) increased the potency about twofold in PJ-32 and PJ-12 relative to the respective original peptides PJ-22 and PJ-1 (Table 5 and Dataset S1). This represented the only identified improvement of potency in the R3 substituent series.
Correlation between physicochemical properties of R3 substituents and the potency of resulting peptides revealed that lipophilic moieties were tolerated better than hydrophilic ones (SI Appendix, Figs. S10 A and D and S11 A and D). Potency also improved with increasing bulk of substituents, reaching optimum between 100 and 120 Å3, further depending on the shape of these groups (SI Appendix, Figs. S10 B and C and S11 B and C). Thus, adding an extra methyl group to the tert-butyl (PJ-33) did not improve the activity while removing one (PJ-18) led to about 50-fold loss in potency; removing two (PJ-17) or three (PJ-16) methyl groups produced inactive compounds (Table 5). Several other variations of the alkyl side chain (PJs 19, 28, and 36) were detrimental except for PJ-26 that was about fourfold more potent than PJ-22 (Table 5).
We next examined whether any of the modified PJs might induce dimerization of the JHR protein pairs from the species that failed to respond to PJ-1 (Fig. 3). When systematically tested in two-hybrid assays, only 13 of the new PJs active on P. apterus showed a detectable effect on JHR from T. castaneum, P. simplex, and D. melanogaster. However, to elicit appreciable cross-species activity, these compounds, including the most potent PJ-105, required concentrations three to five orders of magnitude higher relative to the EC50 values determined for P. apterus JHR (Dataset S1). These marginal effects indicate that while our derivatives retain selectivity toward P. apterus, there may be other chemical modifications that would enable PJs to target more species in the future.
Interaction Energy Governs JHR Response to PJs.
The two-hybrid assays and SAR show that the signaling response of P. apterus JHR depends on the structure of the ligand and most likely reflects the binding affinity to the receptor protein Met. The Per/Arnt/Sim-B (PAS-B) domain of Met is essential for ligand response, and previous modeling suggested that the internal cavity constitutes the ligand-binding site (13, 18, 28). This is analogous to agonist binding to the hypoxia-inducible factor 2 HIF-2 (48), which was identified as the best ligated template for modeling by SWISS-MODEL. The resulting structural model for PaMet PAS-B served as a starting point for insertion of selected ligands. SI Appendix, Fig. S13 A–D illustrates the process using insertion of the native ligand, JHSB3. Note that in the absence of high-resolution structure the elongated nature of the binding cavity required that we consider two initial orientations of the asymmetric ligand as shown in SI Appendix, Fig. S13B. This circumvented the limited sampling of the configuration space provided by subsequent molecular dynamics (MD) simulations which are unlikely to yield ligand reorientation or exit and reentry in a computationally tractable time. In the case of JHSB3 the two starting configurations led to different outcomes after MD equilibration. The configuration with the hydrophobic dimethyl end placed deep into the cavity produced a fully buried ligand configuration (SI Appendix, Fig. S13C). In contrast, the relatively polar methyl ester end, when placed deep into the cavity, tended to drive the ligand out of the cavity (SI Appendix, Fig. S13D). While both structures reached a stationary point as judged by stable Root-Mean-Square Deviation (RMSD) (SI Appendix, Fig. S13E), the former eventually converged to a lower ligand interaction energy, computed using the Generalized Born Surface Area approximation (SI Appendix, Materials and Methods and Fig. S13F) and is considered the correct binding configuration. However, other ligands, such as methoprene, did not exhibit clear orientational preference and yielded similar interaction energies, Eint = −(143 ± 11) and −(149 ± 10) kJ/mol, respectively, in both orientations.
The partial exit of JHSB3 (SI Appendix, Fig. S13D) suggests that the gap between the so-called gate helix and an N-terminal lid loop (SI Appendix, Fig. S13B) is the preferred site for ligand entry and exit from the internal cavity and that it acts as an expandable gate. We further explored this idea by introducing the above-described mutation T274Y (Fig. 2B) within the binding cavity. The bulky tyrosine residue has been shown to disrupt JH signaling by disabling ligand binding (13, 18, 27, 44). Simulation of this mutant complexed with methoprene showed that the mutation caused gradual exit of the ligand through the gate (SI Appendix, Fig. S14). Furthermore, this result demonstrated predictive value of the structural model and suggested that ligand affinity governs the biological response. If this is the case, then one would expect a linear relationship between the log(EC50) of the response and the average ligand interaction energy. Indeed, such a relationship was observed for selected peptidic ligands (Fig. 5). Similar relationships were obtained for various ligands and the crustacean orthologs of Met (49), suggesting that the biological response of JHR is universally determined by ligand affinity.
Fig. 5.
Structural Changes Associated with Ligand Binding.
Simple visual comparison between representative frames of the empty and complexed PaMet PAS-B (SI Appendix, Fig. S13) suggests that conformational changes localize to the gate helix and lid loop. To identify the affected regions and pinpoint the conformational changes, we employed ensemble level analyses of the MD trajectories. First, we computed residue-specific root mean square fluctuations (RMSF) for the core domain of PAS-B excluding the wildly fluctuating N- and C-terminal inter-domain linkers in the empty and JHSB3-bound states (SI Appendix, Materials and Methods and Fig. S15). While the C-terminal β-sheet remained invariant, the fluctuations of the gate helix and lid loop were reduced by ligand binding.
To further pinpoint the conformational changes associated with ligand binding, we removed the ligand and equilibrated the conformational ensemble by long MD. After reaching a stationary state, we concatenated the complex and empty trajectories and subjected this ensemble to dimensionality reduction by principal component analysis (50). As expected, the resulting principal components were dominated by a single coordinate that represented about 80% of all variation. Cluster analysis identified two conformational ensembles representing the complex and the ligand-free state, respectively (SI Appendix, Fig. S16A). The representative median frames from each population are compared in SI Appendix, Fig. S16B, and the variation along the dominant principal component is further illustrated in Movie S1. As shown through cluster analysis, the ligand-bound ensemble conformations were confined to a tighter basin (SI Appendix, Fig. S16A, lower dispersion along Principle Component 2; PC2), i.e., it was less dynamic. In accord with the RMSF analysis, the lid loop and gate helix were the two structural elements that changed conformation upon ligand binding. Thus, the relative position of these two structural motifs and ordering of the gate helix may constitute the structural signature that is necessary for Met domain rearrangement that leads to binding Tai.
Movie S1
Structural changes in the PAS-B domain of PaMet associated with ligand binding. Movie illustrates conformational change (magenta) along the principal component 1 (Fig. S16A) from complex (cyan) to empty (wheat) PAS-B core. Bound JHSB3 is represented as space fill in CPK colors.
Comparison of the Met PAS-B bound to the native ligand (JHSB3) with that bound to the most potent peptidic agonist PJ-105 provided further insight into the increased affinity (Fig. 6A). While both ligand-bound conformations shared a well-organized gate helix, the actual gap was larger for the peptidic ligand. This accommodated the bulky R2 and R3 substituents, effectively creating more contacts with the hydrophobic facet of the gate helix. The more flexible JHSB3 set deeper in the cavity, thus allowing the gate helix to close.
Fig. 6.
Comparing the cavity size was somewhat surprising: PJ-105, with its bulky substituents, yielded a cavity (655 Å3) smaller than the slim but flexible JHSB3 (783 Å3). However, this is consistent with SAR analysis and binding energy estimates (dominated by the extent of buried surface and van der Waals interactions), which both suggest that optimal cavity filling is the key factor determining affinity. The optimal cavity filling led to roughly twice as many side chains with van der Waals contacts with PJ-105 than the native ligand JHSB3 (Fig. 6 B and C). The dynamic nature of the gate helix and of the lid clearly plays a role in accommodating the high-affinity peptidic ligands with bulky substituents. On the other hand, the relatively rigid β-sheet “backbone” constitutes the structurally invariant core and thus cannot adjust around slim ligands such as methoprene or JHSB3 but provides enough space for the R2 substituents. The available space between the β-sheet “backbone” and the gate helix then sets the limit to the volume of substituents at R2 and R3 positions, while the length at position R1 is limited by the depth of the cavity, both of which are finely tunable by the position of the gate helix.
Conclusions
Discovered five decades ago, peptidic juvenoids have remained the most striking example of species selectivity among synthetic mimics of insect JHs. Yet the mode of action of these unique and highly potent compounds has not been explored. This study addresses the interaction of PJs with the JHR of the P. apterus bug, a species exquisitely sensitive to these compounds. Using RNAi-mediated knockdown of the JHR Met, we show that Met is required for the peptidic juvenoid to prevent metamorphosis of larvae to adults. At the molecular level, active PJs specifically induce ligand-dependent interaction between the JHR complex proteins Met and Tai from P. apterus but not from insects that are insensitive to PJ action. We have adapted a two-hybrid assay as well as a NanoBiT split-luciferase system allowing us to monitor Met–Tai interaction in real time. Chemical modifications of the N- and C-terminal side chains and the central carbon atom in the peptidic scaffold yielded more than one hundred PJs which we synthesized. Highly quantitative data from the two-hybrid assay enabled activity ranking and analysis revealing the SAR principles governing the biological activity of these peptides. Strikingly, many of our new PJs surpassed the potency of the original analog, ethyl pivaloyl-l-alanyl-p-aminobenzoate, the best ones by two orders of magnitude. Importantly, the potencies of the PJs inferred from the cell-based interaction assay correlated with their biological anti-metamorphic activity and ability to provoke oogenesis in reproductively diapausing P. apterus females. We further employed molecular modeling in attempt to unveil details of the receptor–agonist interaction. Energies of ligand binding, calculated from the model, correlated with the agonist potencies determined in the two-hybrid assay. The modeling has also identified a putative entry/exit site to the ligand-binding cavity of Met and suggested structural changes occurring during the receptor–agonist interaction. Together, the data presented in this study establish the PJs as genuine agonists of the P. apterus JHR and suggest that their high potency relates to the optimal filling of the binding cavity, which is achieved by exploitation of the binding site flexibility. Despite modifications, the PJs have thus far retained their former selectivity to P. apterus. We anticipate that the new insight afforded by molecular modeling and SAR will present an exciting starting point for future design of similar peptidic JHR agonists that would selectively target insect pests and disease vectors.
Materials and Methods
Compounds and Chemical Synthesis.
10R,S-JH III was from Sigma-Aldrich. (2R,3S)-3-[(3E)-6-[(2R)-3,3-dimethyl-2-oxiranyl]-4-methyl-3-hexen-1-yl]-3-methyl-2-oxiranecarboxylic acid methyl ester (JHSB3) (Toronto Research Chemicals; J211030) was kindly provided by Dr. F. Noriega; purity of this JHSB3 preparation was estimated at 65% based on Gass Chromatography-Mass Spectrometry (GC-MS) analysis (SI Appendix, Fig. S4). JH I [10R,11S-(2E,6E)-JH I] was described earlier (28). The JHs were diluted in ethanol. The synthetic juvenoids, fenoxycarb (Sigma-Aldrich), and (S)-methoprene (VUOS Pardubice, Czechia) were dissolved in dimethylsufoxid (DMSO). Peptidic juvenoids were synthesized at the Institute of Organic Chemistry and Biochemistry (Prague, Czechia) as described in SI Appendix, Materials and Methods, where the chemical structures, MS, and NMR data for all 121 synthesized PJ compounds are provided. The peptides were dissolved and stored in DMSO; for in vivo application, they were further diluted in acetone.
Insects and Juvenoid Treatments.
P. apterus cultures (strain Oldrichovec) were supplied with linden seeds and water and reared at 25°C under long-day photoperiod of 18 h light/6 h darkness (reproductive conditions). To assess effects on metamorphosis, final (L5) instar larvae no older than 24 h after the last ecdysis were separated from the culture, anesthetized under CO2, and treated topically on dorsal abdomen with 3 µl of a tested compound in acetone or the vehicle alone (37). Phenotype of treated animals was scored 10–12 d later. For Kr-h1 mRNA quantification, larvae were sacrificed 6 h post-treatment. To test for effects on inducing vitellogenesis in reproductively diapausing adult females, P. apterus cultures were kept at 25°C under short-day photoperiod of 12 h light/12 h darkness (diapause conditions) starting from the penultimate larval instar. Female adults aged 5 d were treated with 5 µl of tested compounds or acetone alone as described above. Five days later, vitellogenic ovaries developing in treated females under the diapause conditions were scored upon dissection (37).
RNAi.
Knockdown of Met in P. apterus larvae prior to juvenoid treatment was achieved by injecting dsRNA prepared as described previously (36). Late penultimate (L4) instar larvae received 2 µg Met dsRNA in 1 µl water per larva. Within 2–3 h after ecdysis to the L5 instar, the larvae were treated with 0.5 µg of either methoprene, peptide juvenoid (PJ-1), or acetone alone, and their phenotype was determined as described above.
Reverse Transcription-Quantitative Polymarese Chain Reaction (RT-qPCR).
Total RNA was isolated from whole L5 P. apterus larvae using the Trizol reagent (Invitrogen) and treated with Turbo DNase (Ambion) before phenol/chloroform extraction and ethanol precipitation. SuperScript III reverse transcriptase (Invitrogen) and oligo(dT) primers were used to produce cDNA from 2-µg RNA aliquots. Quantitative PCR was performed using the CFX Connect Real-Time System (Bio-Rad) with 2x SYBR Master Mix (Top-Bio) as previously described (39). Primer sequences are listed in SI Appendix, Table S1.
NanoBiT Protein–Protein Interaction System.
DNA sequence encoding the entire Met protein from P. apterus (PaMet; NCBI Accession ON009027) was optimized for human codon usage and custom synthesized (GenScript). A cDNA encoding amino acids M1-P507 of the PaTai protein (NCBI Accession ON009028) was obtained from total RNA of P. apterus L5 larvae by RT-PCR and verified by sequencing. The sequences for PaMet and PaTai were both cloned to each of the pBiT1.1-N [TK/LgBiT] and pBiT2.1-N [TK/SmBiT] vectors (Promega) for expression of the proteins with N-terminally attached large and small fragments of the NanoLuc luciferase, respectively. While both combinations were functional, the one with SmBiT-PaMet and LgBiT-PaTai was used in most experiments. Similar constructs were prepared for expression of the entire T. castaneum Met (TcMet; NCBI Accession NP_001092812.1) and amino acid sequence M1-V505 of TcTai (NCBI Accession XP_008193629.1). Tai orthologs of both species were truncated to contain the N-terminal bHLH and both PAS domains, required for dimerization with Met (13, 15).
For the assay, CHO cells were seeded semiconfluent on solid white, 96-well plates (#3917, Corning), and 24 h later transfected with 50 ng of each expression plasmid per well using 0.3 µl FuGENE HD (Promega). After another 24 h, cells were equilibrated for 15 min in serum-free Dulbecco's Modified Eagle's Medium (DMEM) containing 20 mM 2-[4-(2-Hydroxyethyl)piperazin-1-yl]ethane-1-sulfonic acid (HEPES; pH 7.2), and the Nano-Glo Live Cell Substrate (Promega) was added according to the manufacturer’s instructions. Tested compounds or solvent controls were added, and luminescence was monitored for 20 min using the Orion II (Berthold Detection Systems) microplate reader. For each well and time point, the level of dimerization was determined as fold increase of measured signal relative to baseline luminescence.
Two-Hybrid Assay.
To express the entire PaMet and truncated PaTai (M1-V508) as N-terminal fusion proteins with the VP16 transactivation domain or the Gal4 DNA-binding domain, respectively, the above-described P. apterus sequences were cloned to the pACT and pBIND vectors (Promega). Substitution of individual amino acids (W272A and T274Y) within the ligand-binding pocket was achieved through site-directed mutagenesis of the DNA encoding wild-type (WT) PaMet using PCR with primers shown in SI Appendix, Table S2. Expression of the WT and mutated PaMet proteins in CHO cells was verified by immuno-blotting with a rabbit anti-VP16 antibody (sc-7545, Santa Cruz Biotechnology; 1:1000). pACT and pBIND vectors expressing JHR proteins from species other than P. apterus were kindly provided by Dr. H. Miyakawa (40) or described recently (44).
Agonist-stimulated interaction between Met and Tai proteins was assessed as described (40) with modifications (28). Briefly, CHO cells in 24-well plates were transiently co-transfected using FuGENE HD (Promega) with the appropriate pACT-Met and pBIND-Tai constructs (each 110 ng DNA/well) and 220 ng/well of the pG5luc reporter plasmid (Promega). At 24 h post-transfection, control or tested compounds were added. Cells were incubated for another 20 h, and the firefly luciferase activity, normalized against a Renilla reference, was determined using the Dual-Luciferase system (Promega). EC50 values were calculated using Prism 6.0 GraphPad Software (San Diego, CA) by nonlinear regression (least squares ordinary fit) with the “sigmoidal dose-response (variable slope)” function.
High-Throughput Determination of Agonist Potencies.
To ensure high reproducibility of the automated procedure, the above-described two-hybrid assay was modified. First, the pG5luc reporter was replaced with a pNLCoI-UAS plasmid, prepared by inserting nine copies of the upstream activating sequence (UAS) into the pNLCoI1[luc2-P2A-NlucP/Hygro] coincidence reporter (Promega), which permits detection of signals from the firefly and NanoLuc luciferases. Second, PaTai (M1-V508) fused to the Gal4 DNA-binding domain (DBD) was expressed from a pDBD vector based on the pcDNA3.1/Zeo(+) plasmid backbone (44). The pACT-PaMet vector bearing neomycin resistance was as described above. The three types of antibiotic resistance conferred by the expression and reporter plasmids enabled selection of a stable U2OS cell line carrying all three constructs. From these cells, clones were isolated and screened for optimal response (inducibility and reproducibility) to reference agonists. Resulting clonal, stably transfected cells were used in automated determination of EC50 values for all tested PJs as detailed in SI Appendix, Materials and Methods. Briefly, the cells were added to 384-well plates containing tested compounds, pre-dispensed using contactless acoustic transfer (ECHO 550, Labcyte) at multiple concentration points. Firefly and NanoLuc activities were measured 12 h later using the PHERAstar FSX plate reader (BMG LABTECH) with the Nano-Glo Dual-Luciferase Reporter Assay System (Promega), while cell viability was monitored in parallel using the CellTiter-Glo (Promega) assay. All data were processed using a proprietary LIMS system ScreenX.
Molecular Modeling.
Details on homology modeling for ligand binding, MD simulations, and computation of interaction energy and structural changes are described in SI Appendix, Materials and Methods.
Data, Materials, and Software Availability
All study data are included in the article and/or SI Appendix.
Acknowledgments
We highly appreciate help of Olga Martinkova and Michaela Lisova in cell culturing and preparation of stable cell lines; Martin Popr and Tomas Langhammer helped with compound management and laboratory automation, respectively. We are grateful to Vlastimil Smykal for providing complete P. apterus DNA sequences and advice on reproductive diapause and to Hanka Vaneckova for sharing P. apterus culture. We thank Fernando Noriega and Karel Slama for generous gifts of JHSB3 and JH I, respectively, and Natan Horacek for GC-MS of JHSB3. This research was supported by grant project 20-05151X from the Czech Science Foundation. Work at the National Infrastructure for Chemical Biology CZ-OPENSCREEN was financed by project No. LM2018130 from the Czech Ministry of Education, Youth, and Sports (MEYS). R.T. and D.R. and all computational resources were supported by ERDF project CZ.02.1.01/0.0/0.0/15_003/0000441.
Author contributions
S.T., I.Š., R.T., P.M., D.S., and M.J. designed research; S.T., M. Milacek, I.Š., M. Muthu, R.T., D.R., P.J., L.B., A.N., D.S., and M.J. performed research; S.T., I.Š., R.T., and D.R. contributed new reagents/analytic tools; S.T., M. Milacek, R.T., P.M., D.S., and M.J. analyzed data; and S.T., I.Š., R.T., P.M., D.S., and M.J. wrote the paper.
Competing interests
The authors declare no competing interest.
Supporting Information
Appendix 01 (PDF)
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Dataset S01 (XLSX)
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Movie S1
Structural changes in the PAS-B domain of PaMet associated with ligand binding. Movie illustrates conformational change (magenta) along the principal component 1 (Fig. S16A) from complex (cyan) to empty (wheat) PAS-B core. Bound JHSB3 is represented as space fill in CPK colors.
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Copyright © 2022 the Author(s). Published by PNAS. This article is distributed under Creative Commons Attribution-NonCommercial-NoDerivatives License 4.0 (CC BY-NC-ND).
Data, Materials, and Software Availability
All study data are included in the article and/or SI Appendix.
Submission history
Received: September 15, 2022
Accepted: October 20, 2022
Published online: November 22, 2022
Published in issue: November 29, 2022
Change history
November 23, 2022: Due to a production error, Tables 2 and 5 appeared incorrectly; these tables has been updated. Author Ivan Šnajdr’s name has also been updated to correct a typographical error.Previous version (November 22, 2022)
Keywords
Acknowledgments
We highly appreciate help of Olga Martinkova and Michaela Lisova in cell culturing and preparation of stable cell lines; Martin Popr and Tomas Langhammer helped with compound management and laboratory automation, respectively. We are grateful to Vlastimil Smykal for providing complete P. apterus DNA sequences and advice on reproductive diapause and to Hanka Vaneckova for sharing P. apterus culture. We thank Fernando Noriega and Karel Slama for generous gifts of JHSB3 and JH I, respectively, and Natan Horacek for GC-MS of JHSB3. This research was supported by grant project 20-05151X from the Czech Science Foundation. Work at the National Infrastructure for Chemical Biology CZ-OPENSCREEN was financed by project No. LM2018130 from the Czech Ministry of Education, Youth, and Sports (MEYS). R.T. and D.R. and all computational resources were supported by ERDF project CZ.02.1.01/0.0/0.0/15_003/0000441.
Author contributions
S.T., I.Š., R.T., P.M., D.S., and M.J. designed research; S.T., M. Milacek, I.Š., M. Muthu, R.T., D.R., P.J., L.B., A.N., D.S., and M.J. performed research; S.T., I.Š., R.T., and D.R. contributed new reagents/analytic tools; S.T., M. Milacek, R.T., P.M., D.S., and M.J. analyzed data; and S.T., I.Š., R.T., P.M., D.S., and M.J. wrote the paper.
Competing interests
The authors declare no competing interest.
Notes
This article is a PNAS Direct Submission.
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